CN106754704B - Method for inducing and expanding immune cells in vitro - Google Patents
Method for inducing and expanding immune cells in vitro Download PDFInfo
- Publication number
- CN106754704B CN106754704B CN201611233042.5A CN201611233042A CN106754704B CN 106754704 B CN106754704 B CN 106754704B CN 201611233042 A CN201611233042 A CN 201611233042A CN 106754704 B CN106754704 B CN 106754704B
- Authority
- CN
- China
- Prior art keywords
- cells
- immune
- medium
- immune cell
- culture
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 210000002865 immune cell Anatomy 0.000 title claims abstract description 125
- 238000000034 method Methods 0.000 title claims abstract description 35
- 238000000338 in vitro Methods 0.000 title claims abstract description 14
- 230000001939 inductive effect Effects 0.000 title claims abstract description 11
- 230000003321 amplification Effects 0.000 claims abstract description 61
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 61
- 210000004027 cell Anatomy 0.000 claims abstract description 54
- 239000002609 medium Substances 0.000 claims abstract description 45
- 239000001963 growth medium Substances 0.000 claims abstract description 43
- 230000005931 immune cell recruitment Effects 0.000 claims abstract description 30
- 101000917858 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor III-A Proteins 0.000 claims abstract description 5
- 101000917839 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor III-B Proteins 0.000 claims abstract description 5
- 102100029185 Low affinity immunoglobulin gamma Fc region receptor III-B Human genes 0.000 claims abstract description 5
- 239000011248 coating agent Substances 0.000 claims abstract description 5
- 238000000576 coating method Methods 0.000 claims abstract description 5
- 210000000822 natural killer cell Anatomy 0.000 claims description 42
- 210000004698 lymphocyte Anatomy 0.000 claims description 24
- 108010002350 Interleukin-2 Proteins 0.000 claims description 18
- 102000000588 Interleukin-2 Human genes 0.000 claims description 18
- 230000010261 cell growth Effects 0.000 claims description 9
- 101000581981 Homo sapiens Neural cell adhesion molecule 1 Proteins 0.000 claims description 6
- 102100027347 Neural cell adhesion molecule 1 Human genes 0.000 claims description 6
- 238000013341 scale-up Methods 0.000 claims description 6
- 239000012679 serum free medium Substances 0.000 claims description 4
- 230000004913 activation Effects 0.000 claims description 3
- 239000004017 serum-free culture medium Substances 0.000 claims description 3
- IQWCBYSUUOFOMF-QTLFRQQHSA-N sabcomeline Chemical compound C1CC2[C@@H](C(/C#N)=N/OC)CN1CC2 IQWCBYSUUOFOMF-QTLFRQQHSA-N 0.000 claims 1
- 229950000425 sabcomeline Drugs 0.000 claims 1
- 230000006698 induction Effects 0.000 abstract description 14
- 210000005087 mononuclear cell Anatomy 0.000 abstract description 12
- 238000011282 treatment Methods 0.000 abstract description 11
- 230000008901 benefit Effects 0.000 abstract description 6
- 210000002381 plasma Anatomy 0.000 description 25
- 206010028980 Neoplasm Diseases 0.000 description 21
- 210000005259 peripheral blood Anatomy 0.000 description 13
- 239000011886 peripheral blood Substances 0.000 description 13
- 230000012010 growth Effects 0.000 description 11
- 210000004881 tumor cell Anatomy 0.000 description 11
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 10
- 239000000047 product Substances 0.000 description 10
- 201000011510 cancer Diseases 0.000 description 9
- 210000004369 blood Anatomy 0.000 description 8
- 239000008280 blood Substances 0.000 description 8
- 238000001514 detection method Methods 0.000 description 8
- 230000036737 immune function Effects 0.000 description 8
- 230000001976 improved effect Effects 0.000 description 8
- 230000035755 proliferation Effects 0.000 description 8
- 239000006285 cell suspension Substances 0.000 description 7
- 230000036039 immunity Effects 0.000 description 7
- 208000015181 infectious disease Diseases 0.000 description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 241000700605 Viruses Species 0.000 description 6
- 238000012258 culturing Methods 0.000 description 6
- 230000034994 death Effects 0.000 description 6
- 231100000517 death Toxicity 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- 230000006872 improvement Effects 0.000 description 6
- 239000002504 physiological saline solution Substances 0.000 description 6
- 230000008569 process Effects 0.000 description 6
- 241000204031 Mycoplasma Species 0.000 description 5
- 239000000427 antigen Substances 0.000 description 5
- 102000036639 antigens Human genes 0.000 description 5
- 108091007433 antigens Proteins 0.000 description 5
- 238000003556 assay Methods 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 239000002158 endotoxin Substances 0.000 description 5
- 230000001965 increasing effect Effects 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 241000699670 Mus sp. Species 0.000 description 4
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 4
- 230000036541 health Effects 0.000 description 4
- 210000002443 helper t lymphocyte Anatomy 0.000 description 4
- 238000011580 nude mouse model Methods 0.000 description 4
- 210000005105 peripheral blood lymphocyte Anatomy 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- 102100037850 Interferon gamma Human genes 0.000 description 3
- 108010074328 Interferon-gamma Proteins 0.000 description 3
- KHGNFPUMBJSZSM-UHFFFAOYSA-N Perforine Natural products COC1=C2CCC(O)C(CCC(C)(C)O)(OC)C2=NC2=C1C=CO2 KHGNFPUMBJSZSM-UHFFFAOYSA-N 0.000 description 3
- 229930188599 Sapelin Natural products 0.000 description 3
- 210000001744 T-lymphocyte Anatomy 0.000 description 3
- 230000001093 anti-cancer Effects 0.000 description 3
- 239000006143 cell culture medium Substances 0.000 description 3
- 230000003833 cell viability Effects 0.000 description 3
- 239000012228 culture supernatant Substances 0.000 description 3
- 210000004748 cultured cell Anatomy 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 238000009169 immunotherapy Methods 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 229930192851 perforin Natural products 0.000 description 3
- 230000028327 secretion Effects 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 231100000588 tumorigenic Toxicity 0.000 description 3
- 230000000381 tumorigenic effect Effects 0.000 description 3
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 2
- 102000004127 Cytokines Human genes 0.000 description 2
- 108090000695 Cytokines Proteins 0.000 description 2
- 241000725303 Human immunodeficiency virus Species 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 241000699660 Mus musculus Species 0.000 description 2
- 241000589884 Treponema pallidum Species 0.000 description 2
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 2
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 2
- 210000003719 b-lymphocyte Anatomy 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 235000015895 biscuits Nutrition 0.000 description 2
- 210000000601 blood cell Anatomy 0.000 description 2
- 210000001185 bone marrow Anatomy 0.000 description 2
- 230000004663 cell proliferation Effects 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 230000007123 defense Effects 0.000 description 2
- 210000003743 erythrocyte Anatomy 0.000 description 2
- 230000005714 functional activity Effects 0.000 description 2
- 208000002672 hepatitis B Diseases 0.000 description 2
- 230000002147 killing effect Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 210000002540 macrophage Anatomy 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 230000037361 pathway Effects 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- GOZMBJCYMQQACI-UHFFFAOYSA-N 6,7-dimethyl-3-[[methyl-[2-[methyl-[[1-[3-(trifluoromethyl)phenyl]indol-3-yl]methyl]amino]ethyl]amino]methyl]chromen-4-one;dihydrochloride Chemical compound Cl.Cl.C=1OC2=CC(C)=C(C)C=C2C(=O)C=1CN(C)CCN(C)CC(C1=CC=CC=C11)=CN1C1=CC=CC(C(F)(F)F)=C1 GOZMBJCYMQQACI-UHFFFAOYSA-N 0.000 description 1
- 238000011729 BALB/c nude mouse Methods 0.000 description 1
- 102000019034 Chemokines Human genes 0.000 description 1
- 108010012236 Chemokines Proteins 0.000 description 1
- 206010053567 Coagulopathies Diseases 0.000 description 1
- 244000078127 Eleusine coracana Species 0.000 description 1
- 235000013499 Eleusine coracana subsp coracana Nutrition 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 206010018910 Haemolysis Diseases 0.000 description 1
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 description 1
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 description 1
- 102000008100 Human Serum Albumin Human genes 0.000 description 1
- 108091006905 Human Serum Albumin Proteins 0.000 description 1
- 229920001612 Hydroxyethyl starch Polymers 0.000 description 1
- 108010002352 Interleukin-1 Proteins 0.000 description 1
- 102000004503 Perforin Human genes 0.000 description 1
- 108010056995 Perforin Proteins 0.000 description 1
- 206010070834 Sensitisation Diseases 0.000 description 1
- 241000973497 Siphonognathus argyrophanes Species 0.000 description 1
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000033289 adaptive immune response Effects 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 229940124650 anti-cancer therapies Drugs 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 238000011319 anticancer therapy Methods 0.000 description 1
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 229940127219 anticoagulant drug Drugs 0.000 description 1
- 229960002233 benzalkonium bromide Drugs 0.000 description 1
- KHSLHYAUZSPBIU-UHFFFAOYSA-M benzododecinium bromide Chemical compound [Br-].CCCCCCCCCCCC[N+](C)(C)CC1=CC=CC=C1 KHSLHYAUZSPBIU-UHFFFAOYSA-M 0.000 description 1
- 229940126587 biotherapeutics Drugs 0.000 description 1
- 238000001815 biotherapy Methods 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000035602 clotting Effects 0.000 description 1
- QTCANKDTWWSCMR-UHFFFAOYSA-N costic aldehyde Natural products C1CCC(=C)C2CC(C(=C)C=O)CCC21C QTCANKDTWWSCMR-UHFFFAOYSA-N 0.000 description 1
- 210000004405 cytokine-induced killer cell Anatomy 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 230000002354 daily effect Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 239000003651 drinking water Substances 0.000 description 1
- 235000020188 drinking water Nutrition 0.000 description 1
- 238000009261 endocrine therapy Methods 0.000 description 1
- 229940034984 endocrine therapy antineoplastic and immunomodulating agent Drugs 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000008588 hemolysis Effects 0.000 description 1
- 210000005104 human peripheral blood lymphocyte Anatomy 0.000 description 1
- 229940050526 hydroxyethylstarch Drugs 0.000 description 1
- 230000007124 immune defense Effects 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 229960003444 immunosuppressant agent Drugs 0.000 description 1
- 230000001861 immunosuppressant effect Effects 0.000 description 1
- 239000003018 immunosuppressive agent Substances 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- ISTFUJWTQAMRGA-UHFFFAOYSA-N iso-beta-costal Natural products C1C(C(=C)C=O)CCC2(C)CCCC(C)=C21 ISTFUJWTQAMRGA-UHFFFAOYSA-N 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 210000003810 lymphokine-activated killer cell Anatomy 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004719 natural immunity Effects 0.000 description 1
- 230000024121 nodulation Effects 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- OXNIZHLAWKMVMX-UHFFFAOYSA-N picric acid Chemical compound OC1=C([N+]([O-])=O)C=C([N+]([O-])=O)C=C1[N+]([O-])=O OXNIZHLAWKMVMX-UHFFFAOYSA-N 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000011127 radiochemotherapy Methods 0.000 description 1
- 235000002079 ragi Nutrition 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 230000008313 sensitization Effects 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 230000005740 tumor formation Effects 0.000 description 1
- 230000005909 tumor killing Effects 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0646—Natural killers cells [NK], NKT cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0018—Culture media for cell or tissue culture
- C12N5/0037—Serum-free medium, which may still contain naturally-sourced components
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/06—Anti-neoplasic drugs, anti-retroviral drugs, e.g. azacytidine, cyclophosphamide
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
- C12N2501/2302—Interleukin-2 (IL-2)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/50—Cell markers; Cell surface determinants
- C12N2501/599—Cell markers; Cell surface determinants with CD designations not provided for elsewhere
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Hematology (AREA)
- Immunology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention discloses a method for inducing and amplifying immune cells in vitro. The method comprises the following steps: coating the culture container with a CD16 antibody to obtain a coated culture container; placing the single nuclear cells in the coated culture container by using an immune cell activation medium to perform the immune cell activation medium so as to obtain activated immune cells; performing a second induced amplification culture on the immune cells subjected to the primary induced amplification by using an immune cell amplification culture medium so as to obtain amplified immune cells; and performing third induced amplification culture on the differentiated immune cells by using an immune cell scale amplification culture medium so as to obtain scale-amplified functionally activated immune cells. The method is used for carrying out induction amplification culture on the mononuclear cells to obtain large-scale immune cells, and has the advantages of high induction efficiency, high amplification speed, high safety, low cost and the like, thereby meeting the requirement of clinical treatment of a large number of immune cells.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for inducing and amplifying immune cells in vitro.
Background
In recent years, biotherapy has become the fifth treatment mode following surgery, chemoradiotherapy and endocrine therapy, and has been increasingly emphasized. Adoptive Cellular Immunotherapy (ACI) is one of the cell biotherapeutics, and it is to infuse immune cells with anti-tumor activity into tumor patients to directly kill tumor cells or stimulate the immune response of the body to kill tumor cells, so as to achieve the purpose of treating tumors. At present, adoptive immune cells clinically applied comprise DC-CIK cells, TIL cells, LAK cells and NK cells, wherein the CIK cells, the LAK cells and the A-NK cells are all anti-cancer systems taking natural killer cells as main bodies.
Natural killer cells (natural killer cells) also called NK cells, mainly derived from bone marrow CD34+The NK cells are distributed in bone marrow, peripheral blood and spleen, account for 10% -20% of peripheral blood lymphocytes, the NK cells play an important role in tumor immunity, clearing non-hexylic cells and the like, are the main component of natural immune defense, are positioned at the first defense line of a body defense system, have no MHC restriction on the killing activity of the NK cells, do not rely on antibodies, can identify and kill tumor and virus-infected cells without antigen pre-sensitization, directly play a cytotoxic role in the tumor cells through a perforin-granzyme pathway and a Fas-FasL pathway, and simultaneously can secrete various cytokines and chemokines such as TNF- α, IFN-gamma and IL-1 and the like in early onset, the cytokines are involved in anti-cancer and regulating acquired immune response, so the NK cells are also important bridges connecting natural immunity with acquired immunity, although the safety and the curative effect of the anti-cancer effect of the NK cells are ensured because the NK cells account for 10% -20% of the peripheral blood lymphocytes, the high-quality cell products are key to the near-NK therapy, can generate the relative stimulation to the in-vitro expansion of the NK cells through the in-2-NK cell proliferation and the in-NK cell proliferation of the IL-cell proliferation, the IL-cell proliferation-cell growth of the IL-cell growth of the near-cell, the NK cell growth of the NK cell, the growth of the human tumor cells can be realized by the growth of the human tumor cells, the human growth of the human tumor cells, the human tumor cells and the human tumor cells, the human growth of the human tumor cells, the human growth of the human tumor cells can be induced by the human growth of the human tumor cells, the human growth of the humanSex, and is dose-dependent. However, the existing NK cell therapeutic products in the current market have the problems of complex culture system, multiple amplification and poor tumor killing effect, and part of the culture system has safety risk and the like.
Thus, methods for culturing natural killer cells are in need of improvement.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art. Therefore, an object of the present invention is to provide a method for inducing and amplifying immune cells in vitro, wherein the method has the advantages of high induction efficiency, high amplification speed, high safety, low cost, etc.
According to one aspect of the invention, there is provided a method of inducing expansion of immune cells in vitro. According to an embodiment of the invention, the immune cell culture medium system comprises: the method comprises the following steps: coating the culture container with a CD16 antibody to obtain a coated culture container; placing the single nuclear cells in the coated culture container by using an immune cell activation medium to perform the immune cell activation medium so as to obtain activated immune cells; performing a second induced amplification culture on the immune cells subjected to the primary induced amplification by using an immune cell amplification culture medium so as to obtain amplified immune cells; performing third induced amplification culture on the differentiated immune cells by using an immune cell large-scale amplification culture medium so as to obtain large-scale amplified functional activated immune cells, wherein the immune cell activation culture medium is a serum-free lymphocyte culture medium added with plasma, interleukin-2 and saperin; the immune cell amplification culture medium is a serum-free lymphocyte culture medium added with interleukin-2 and saperin; and the immune cell scale amplification culture medium is a serum-free lymphocyte culture medium added with interleukin-2.
The inventor surprisingly finds that the method for inducing, amplifying and culturing the mononuclear cells to obtain the large-scale immune cells has the advantages of high induction efficiency, high amplification speed, high safety, low cost and the like, thereby meeting the requirement of clinically treating a large amount of immune cells.
In addition, the method for inducing the expansion of the immune cells in vitro according to the above embodiment of the present invention may further have the following additional technical features:
according to an embodiment of the present invention, the concentration of said sapelin in said immune cell activation medium and said immune cell expansion medium is 0.007-0.013KE/ml, preferably 0.01KE/ml, respectively.
According to the embodiment of the present invention, the concentration of interleukin-2 in the immune cell activation medium, the immune cell expansion medium and the immune cell scale-up expansion medium is 700-1300IU/ml, preferably 1000 IU/ml.
According to the embodiment of the invention, in the immune cell activation medium, the immune cell amplification medium and the immune cell scale amplification medium, the serum-free lymphocyte culture medium is: OpTsizerTMCTSTMSerum-free medium or SupercultureTML500 human lymphocyte serum-free culture medium.
According to an embodiment of the invention, the concentration of said plasma in said immune cell activation medium is 7-13% by volume, preferably 10% by volume.
According to an embodiment of the invention, the plasma is autologous plasma.
According to an embodiment of the invention, the immune cell is a natural killer cell.
According to an embodiment of the invention, the second induced expansion culture is performed when the expression of CD56 by the activated immune cells exceeds 70%.
According to an embodiment of the present invention, the third scale-up induced expansion culture is performed when the expression of CD56 of the differentiated immune cells exceeds 90%.
According to an embodiment of the invention, the immune cells are passaged every 1 day during the activation of the medium.
According to an embodiment of the invention, the second induced expansion culture is passaged every 2 days.
According to an embodiment of the invention, the third induced expansion culture is passaged every 2 days.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:
FIG. 1 is a graph showing the results of culturing peripheral blood mononuclear cell counts and cell viability for various periods of time according to one embodiment of the present invention;
FIG. 2 is a graph showing the results of the proportion of lymphocytes in peripheral blood mononuclear cells before and after 14 days of culture according to an embodiment of the present invention;
FIG. 3 is a graph showing the results of culturing peripheral blood mononuclear cell counts and cell viability for various periods of time according to yet another embodiment of the present invention;
FIG. 4 is a graph showing the results of the proportion of lymphocytes in peripheral blood mononuclear cells before and after 12 days of culture according to an embodiment of the present invention;
FIG. 5 is a graph showing the results of a nude mouse tumorigenic experiment according to an embodiment of the present invention.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the accompanying drawings are illustrative only for the purpose of explaining the present invention, and are not to be construed as limiting the present invention.
It should be noted that the terms "first" and "second" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Further, in the description of the present invention, "a plurality" means two or more unless otherwise specified.
There is a large market for anticancer therapies. International research center for cancer (IARC) reports show that 487 ten thousand new cancer cases and 360 ten thousand death cases are expected in china in 2030, and the market demand for cancer treatment in china will keep increasing. The Ministry of health published "the third national census of death cause" in 2008. Survey data show that the mortality rate of malignant tumors of urban and rural residents in China is at a higher level in the world and is in a continuous increasing trend, and the mortality rate is increased by 83.1 percent and 22.5 percent respectively compared with the mortality rate of the malignant tumors of the 70 th and the 90 th of the last century. The malignant tumor is the first cause of death in cities (accounting for 25.0 percent of the total number of deaths in cities), and the second cause of death in rural areas (accounting for 21.0 percent of the total number of deaths in rural areas). Cancer not only seriously threatens human health, but also is an important factor promoting rapid rise of medical expenses. Taking the united states as an example, the total cost associated with cancer in the united states in 2010 is $ 2638 billion, as assessed by the National Institutes of health. According to data published by the National Cancer Institute (NCI), the direct cost of cancer treatment in the united states in 2010 is $ 1245.7 billion, and this figure is expected to increase to at least $ 1577.7 billion by the year 2020.
NK cells separated from human peripheral blood have good killing effect on various tumors. NK cells obtained by separating and culturing peripheral blood of a patient do not have immunological rejection, and an immunosuppressant does not need to be taken for a long time, so that the life quality of the patient is obviously improved. After the NK cells are back-transfused, the normal body cells can not be attacked, and the health of the human body is not damaged. However, because the number of NK cells in autologous peripheral blood is limited, the NK cells obtained by separating the peripheral blood cannot meet the clinical treatment requirement, and the invention aims to obtain sufficient, safe and effective NK cell products as soon as possible by in vitro amplification culture so as to meet the clinical treatment requirement of tumor patients.
According to one aspect of the invention, there is provided a method of inducing expansion of immune cells in vitro. According to an embodiment of the invention, the immune cell culture medium system comprises: the method comprises the following steps: coating the culture container with a CD16 antibody to obtain a coated culture container; placing the single nuclear cells in the coated culture container by using an immune cell activation medium to perform the immune cell activation medium so as to obtain activated immune cells; performing a second induced amplification culture on the immune cells subjected to the primary induced amplification by using an immune cell amplification culture medium so as to obtain amplified immune cells; performing third induced amplification culture on the differentiated immune cells by using an immune cell large-scale amplification culture medium so as to obtain large-scale amplified functional activated immune cells, wherein the immune cell activation culture medium is a serum-free lymphocyte culture medium added with plasma, interleukin-2 and saperin; the immune cell amplification culture medium is a serum-free lymphocyte culture medium added with interleukin-2 and saperin; and the immune cell scale amplification culture medium is a serum-free lymphocyte culture medium added with interleukin-2.
The inventor surprisingly finds that the method for inducing, amplifying and culturing the mononuclear cells to obtain the large-scale immune cells has the advantages of high induction efficiency, high amplification speed, high safety, low cost and the like, thereby meeting the requirement of clinically treating a large amount of NK cells.
According to a preferred embodiment of the present invention, the concentration of interleukin-2 in the immune cell activation medium and the immune cell expansion medium is 1000 IU/ml. Therefore, the induction and proliferation speed of the immune cells is higher, the amplification efficiency is high, the purity of the obtained immune cells is high, the obtained immune cells have better immune function, and the immune cells can be used for clinical treatment of infection resistance, tumor resistance, immunity improvement and the like.
According to an embodiment of the present invention, the concentration of sapelin in the immune cell activation medium and the immune cell expansion medium is 0.007 to 0.013 KE/ml. Therefore, the induction and proliferation speed of the immune cells is high, the amplification efficiency is high, the purity of the obtained immune cells is high, the obtained immune cells have better immune function, and the immune cells can be used for clinical treatment of infection resistance, tumor resistance, immunity improvement and the like.
According to a preferred embodiment of the present invention, the concentration of sapelin in both the immune cell activation medium and the immune cell expansion medium is 0.01 KE/ml. Therefore, the induction and proliferation speed of the immune cells is higher, the amplification efficiency is high, the purity of the obtained immune cells is high, the obtained immune cells have better immune function, and the immune cells can be used for clinical treatment of infection resistance, tumor resistance, immunity improvement and the like.
According to the embodiment of the invention, the concentration of interleukin-2 in the immune cell activation medium, the immune cell amplification medium and the immune cell scale amplification medium is 700-1300 IU/ml. Therefore, the induction and proliferation speed of the immune cells is high, the amplification efficiency is high, the purity of the obtained immune cells is high, the obtained immune cells have better immune function, and the immune cells can be used for clinical treatment of infection resistance, tumor resistance, immunity improvement and the like.
According to the preferred embodiment of the present invention, the concentration of interleukin-2 in the immune cell activation medium, the immune cell expansion medium and the immune cell scale-up medium is 1000 IU/ml. Therefore, the induction and proliferation speed of the immune cells is higher, the amplification efficiency is high, the purity of the obtained immune cells is high, the obtained immune cells have better immune function, and the immune cells can be used for clinical treatment of infection resistance, tumor resistance, immunity improvement and the like.
According to the embodiment of the invention, in the immune cell activation culture medium, the immune cell amplification culture medium and the immune cell scale amplification culture medium, the serum-free lymphocyte culture medium is: OpTsizerTMCTSTMSerum-free medium or SupercultureTML500 human lymphocyte serum-free culture medium. Therefore, the method is beneficial to the in-vitro efficient amplification culture of immune cells and keeps higher functional activity.
According to an embodiment of the present invention, the concentration of plasma in the immune cell activation medium is 7 to 13 vol%. Therefore, the method is beneficial to the in-vitro efficient amplification culture of immune cells and keeps higher functional activity.
According to a preferred embodiment of the present invention, the concentration of plasma in the immune cell activation medium is 10% by volume. Therefore, the induction and proliferation speed and efficiency of the immune cells are obviously improved, and the obtained immune cells have high purity and better immune function.
An embodiment according to the invention is characterized in that the plasma is autologous plasma. Therefore, the immune cells have small rejection effect on blood plasma, the induction and proliferation speed and efficiency of the immune cells are obviously improved, and the obtained immune cells have high purity and better immune function.
It should be noted that the term "autologous plasma" as used herein refers to plasma from the same source as the mononuclear cells to be induced. In other words, the plasma and the mononuclear cells to be induced and cultured are taken from the same individual.
According to an embodiment of the invention, the immune cell is a natural killer cell. This results in high efficiency of amplification induction.
According to an embodiment of the invention, the second induced expansion culture is performed when the expression of CD56 by said activated immune cells exceeds 70%. Therefore, when most mononuclear cells are induced to differentiate to form immune cells, the second induced amplification culture can be carried out, so that the purity of the immune cells is further improved, the number of the cells is obviously improved, and meanwhile, the function of the immune cells is further enhanced in the amplification process.
According to an embodiment of the present invention, the third scale-up induced expansion culture is performed when the expression of CD56 of the differentiated immune cells exceeds 90%. Therefore, when most mononuclear cells are induced to differentiate to form immune cells, the third induced amplification culture can be carried out, so that the immune cells are further amplified in a large scale, the immune function is maintained, and sufficient cells are provided for possible clinical immunotherapy.
According to an embodiment of the invention, the immune cells are passaged every 1 day during the activation of the medium. Therefore, in the process of activating the culture medium by the immune cells, the immune cells are obviously activated and effectively amplified, and the proportion of other cells is obviously reduced.
According to an embodiment of the invention, the second induced expansion culture is passaged every 2 days. Therefore, in the second induction amplification culture process, the purity of the immune cells is further improved, meanwhile, the proportion of other cells is further reduced, and the function of the immune cells is enhanced.
According to an embodiment of the present invention, the third induced expansion culture is passaged every 2 days. Therefore, in the third induced amplification culture process, the immune cells are amplified in a large scale under high purity, and sufficient functional activated immune cells are obtained for possible clinical immunotherapy.
The present invention is described below with reference to specific examples, which are intended to be illustrative only and are not to be construed as limiting the invention.
The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or apparatus used are conventional products which are commercially available, e.g. from Sigma, without reference to the manufacturer.
Example 1
By using the immune cell culture medium system of the embodiment of the invention, the separated mononuclear cells are induced to be expanded into immune cells, and the activity of the immune cells is detected.
First, experiment method
1. Preparation of anti-human CD 16-coated T75 bottle
1.1 Add 5mL of anti-human CD16 monoclonal antibody dissolved in medical normal saline 2.5. mu.g/mL into a sterile culture flask, gently shake the flask to make the antibody spread over the culture surface, and keep out of the sun overnight at 4 ℃.
1.2 recovery of antibody coating before use, washing of the flask with 5mL of physiological saline, and then 5mL of T cell expansion Medium (OpTsizer)TMCTSTMT-cell expansion SFM) was washed once.
2. Collecting peripheral blood, separating peripheral blood plasma and mononuclear cells
2.1 using the anticoagulant aseptic blood collection bag to collect about 100ml of human peripheral blood, reserving 1ml of peripheral blood for quick test and blood type identification, immediately and aseptically packaging the blood collection bag, storing and transporting at 4 ℃ without bacteria, and accurately recording the collected information. And (5) sending the peripheral blood into a GMP laboratory after the quick test screening is qualified, and if the quick test is unqualified, discarding the blood sample.
2.2 blood bags were removed in the GMP laboratory, alcohol sterilized blood bags, observed for lack of clotting and hemolysis and opened in a clean bench, blood was transferred to a 50ml sterile centrifuge tube (40 ml/tube or less) and centrifuged at 2500rpm for 15 min.
2.3 transferring the upper plasma layer to another sterile centrifuge tube, centrifuging at 3500rpm for 15min, collecting the supernatant plasma to a new sterile centrifuge tube, sealing the centrifuge tube mouth with a mouth membrane, and separating the blood cells from the mononuclear cells.
2.4 putting the plasma into a 56 ℃ water bath kettle for water bath for 30-50min to inactivate the complement, centrifuging at 3500rpm for 15min to remove the complement, subpackaging 10ml of each sample into a 15ml sterile centrifuge tube, and freezing at-20 ℃ for later use; and leave 7.5ml of plasma for slow examination: five viruses, mycoplasma, endotoxin and microorganisms, wherein the virus is detected by a third party.
2.5 the blood cell sediment in step 2 is resuspended by normal saline equal volume of plasma and then transferred to a 250ml sterile glass bottle, hydroxyethyl starch with total volume of 1/3 blood is added, the saline bottle is gently shaken to mix evenly, and the mixture is left to stand to settle the red blood cells.
2.6 after the red blood cell layer is settled and layered, slightly sucking the milky white suspension on the upper layer into a sterile centrifuge tube, centrifuging at 1800rpm for 5min, discarding the supernatant, and re-suspending the precipitate with 10ml of normal saline.
2.7 taking 2 sterile 15ml centrifuge tubes, adding 5ml of normal temperature human peripheral blood lymphocyte separating medium respectively, and adding 5ml of cell suspension respectively on the upper layer. Centrifuge slowly rising and falling at 2000rpm for 25min at room temperature.
2.8 gently take out the centrifuge tube, carefully absorb the middle cloud layer leucocyte in the interface into a new 15ml centrifuge tube, and add physiological saline to wash for 2 times.
2.91 ml of physiological saline was resuspended, 5. mu.l of the cells were added to 245. mu.l of physiological saline to dilute 50-fold, counted, and the cell viability was measured by trypan blue staining. After the experiment is finished, the bloody dirt can be discarded after being soaked overnight in benzalkonium bromide which is prepared by proportioning.
2.10 Reserve 4.5x106Carrying out flow antibody labeling on each cell to detect the proportion of NK cells; the remaining mononuclear cells were inoculatedThe seeds are planted in a culture bottle with 20ml of culture medium, and the mixture ratio of the culture medium is as follows: 18ml OpTsizerTMCTSTMT-celexpansion SFM Medium +2ml Autologous plasma (autorogous plasma) + 1000IU/ml Pepro TechIL-2+ 0.01KE/ml Shapelin (OK432), 37 ℃, 5% CO2Aseptically cultured, and scored as day 0.
2.11 observing cells every day, and carrying out appropriate passage amplification, wherein the proportion of the culture medium is as follows: OpTsizerTMCTSTMT-cell expansion SFM medium + 10% Autologous plasma (autologus plasma) + 1000IU/ml Pepro Tech IL-2 final concentration. When the autologous plasma has been used up, the plasma is not supplemented any more, i.e. OpTsizerTMCTSTMT-cell expansion SFM medium + PeproTech IL-2 at a final concentration of 1000 IU/ml.
2.12 when the culture system has reached 2L, the culture system is replaced, namely SuperCulture TM L500 human lymphocyte serum-free medium + domestic IL-2 with a final concentration of 1000IU/ml, cells are observed daily and appropriately passaged. After 14 days, the cells were recovered and 10ml of the culture supernatant was left for detection of Elisa secretion factor.
2.13 resuspending the recovered cells with 200ml of physiological saline, adding 10ml of human serum albumin, and mixing well; 10ml of cell suspension is taken from the suspension and tested, and the remaining cells are injected into a reinfusion bag to prepare reinfusion. 10ml of cell suspension was withdrawn from the feedback bag for testing: mycoplasma, endotoxin, microorganisms and viruses.
3. Flow cytometry detection of lymphocyte lineages in cells
10ml of the cell suspension was centrifuged at 1800rpm to recover cells, which were labeled with flow antibody, isotype control, single-label sample and staining tube were set, and the number of cells per tube was about 5 × 105Then, corresponding antibody staining was added. Standing at 4 deg.C for 30min, washing with physiological saline, and detecting and analyzing NK cell proportion in lymphocyte population on machine.
4. And (3) subpackaging the residual 10ml of cell suspension supernatant for detecting five virus elements, endotoxin, mycoplasma and microorganisms.
5. Tumor test of nude mice female BALB/c nude mice of SPF grade purchased fromThe research institute of laboratory animals of Chinese academy of medical sciences, 4-6 weeks old, weight 16-20g, raising in covered cage in laminar air flow frame, sterilizing drinking water, standard feed and other animal contacting products, taking positive control Ragi cell and K562 cell and NK cell to be detected on 21 days of in vitro induced differentiation according to 3 × 107Each 0.2ml of the inoculated nude mice is inoculated into the subcutaneous part of the costal area and marked by picric acid, and the nodulation is observed for 2 months.
6. The culture supernatants from the harvested cells were subjected to Elisa secretion factor assays, i.e., IFN-. gamma., TNF-. alpha.and Perforin assays.
Second, experimental results
1 Experimental results of 14 days of culture
1.1 cells can expand 150-fold after 14 days of culture
Separating peripheral blood lymphocyte separation liquid to obtain 6 × 107Inoculating single nuclear cell in peripheral blood into 20ml culture system, making the cell quickly enter logarithmic growth stage, after 14d of culture, expanding culture system to 4L, and expanding cell number to 9 × 109The amplification times are up to 150 times, the number of living cells is over 90%, and the results of the number of mononuclear cells and the cell activity of the peripheral blood cultured for different times are shown in figure 1.
1.2 the proportion of NK cells in peripheral blood mononuclear cells after expansion is obviously increased
NK cell proportion in lymphocytes after expansion of peripheral blood mononuclear cells for 14 days (CD 3)-CD56+) From 18.15% to 95.27%, while T lymphocytes (CD 3)+) The proportion is reduced from 72.15% to 4.61%, and helper T cells (Th, CD 3)+CD4+) And cytotoxic T cells (Tc, CD 3)+CD8a+) All had a decrease, B lymphocytes (CD 3)-CD19+) Basically disappears, and the cell uniformity is obviously improved; as can be seen from the cell count calculation in fig. 1, the NK cells were amplified 780 times after 14 days of culture, and the results of the lymphocyte ratio in peripheral blood mononuclear cells before and after 14 days of culture are shown in fig. 2, in which NK cells: CD3-CD56 +; t cell: CD3 +; helper T cell (Th): CD3+ CD4 +; cytotoxic T cells (Tc cells): CD3+ CD8a +; b cell: CD3-CD9 +.
1.3 detection of cell products obtained by culture amplification of No pathogen infection
The cultured cells and cell suspension are entrusted to a detection platform to detect hepatitis B surface antigen, biscuit antigen, human immunodeficiency virus antibody, treponema pallidum specific antibody, macrophage virus, mycoplasma, bacteria and endotoxin, the detection results are negative, the batch of products are safe, no pollution is caused in the culture process, and the results are detailed in table 1.
TABLE 1 clinical safety assay of NK cells after 14 days of culture
2. Experimental results of 12 days of culture
2.1 cells expanded 75-fold after 12 days of culture
Separating peripheral blood lymphocyte separation liquid to obtain 9 × 107Inoculating single nuclear cell in peripheral blood into 20ml culture system, making the cell quickly enter logarithmic growth stage, after 12d of culture, expanding culture system to 4L, and expanding cell number to 6.76 × 109The amplification fold is 75 times, the number of living cells is more than 90%, and the experimental result is shown in figure 3.
2.2 the proportion of NK cells in peripheral blood mononuclear cells after expansion is significantly increased
NK cell proportion in lymphocytes after 12 days expansion of peripheral blood mononuclear cells (CD 3)-CD56+) From 32.87% on day 7 to 92.19% on day 12, with T lymphocytes (CD 3)+) The proportion drops to 3.24%, helper T cells (Th, CD 3)+CD4+) And cytotoxic T cells (Tc, CD 3)+CD8a+) All had a decrease, B lymphocytes (CD 3)-CD19+) Basically disappeared, cell homogeneity was significantly improved, and the proportion of lymphocytes in peripheral blood mononuclear cells before and after 12 days of culture was detailed in fig. 4, in which NK cells: CD3-CD56 +; t cell: CD3 +; helper T cell (Th): CD3+ CD4 +; cytotoxic T cells (Tc cells): CD3+ CD8a +; b cell: CD3-CD9 +.
2.3 detection of cell products obtained by culture amplification of No pathogen infection
The cultured cells and cell suspension are entrusted to a detection platform to detect hepatitis B surface antigen, biscuit antigen, human immunodeficiency virus antibody, treponema pallidum specific antibody, macrophage virus, mycoplasma, bacteria and endotoxin, the detection results are negative, the batch of products are safe, no pollution is caused in the culture process, and the results are detailed in table 2.
TABLE 2 clinical safety assay of NK cells after 12 days of culture
TABLE 3 culture supernatants for Elisa assay
| Experiment 1 | Experiment 2 | |
| IFN-γ | 1.48x105pg/ml | 1.435x105pg/ml |
| TNF-α | 61.1pg/ml | 48.75pg/ml |
| Perforin | 19ng/ml | 17.48ng/ml |
The cultured cells all have the secretion of IFN-gamma, TNF-alpha and Perforin.
3. The cultured and amplified NK cells cannot form tumors in vivo
The results of the nude mouse tumorigenic experiment are shown in FIG. 5, in which A saline control group (number of tumorigenic mice/number of mice in group, 0/5), B Raji cell control group (3/5), C K562 cell control group (4/5), and D NK cell group (0/5) were injected intradermally with 0.2ml saline and 3 × 10 for 2 months of observation period7No tumor was observed in 0.2ml of mice in the 21-day-old NK cell group, and 3/5 and 4/5 mice injected with Raji cells and K562 cells in the same volume, respectively, had visible tumors. This result indicates that NK cells are safe and effective even when cultured for 21 days, and do not cause tumor formation.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
While embodiments of the invention have been shown and described, it will be understood by those of ordinary skill in the art that: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.
Claims (8)
1. A method for inducing expansion of immune cells in vitro, comprising:
coating the culture container with a CD16 antibody to obtain a coated culture container;
placing the single nuclear cells in the coated culture container by using an immune cell activation medium to perform the immune cell activation medium so as to obtain activated immune cells;
performing a second induced amplification culture on the immune cells subjected to the primary induced amplification by using an immune cell amplification culture medium so as to obtain amplified immune cells; and
performing third induced amplification culture on the differentiated immune cells by using an immune cell scale amplification culture medium so as to obtain scale-amplified functionally activated immune cells,
wherein,
the immune cell activation culture medium is a serum-free lymphocyte culture medium added with plasma, interleukin-2 and saperin;
the immune cell amplification culture medium is a serum-free lymphocyte culture medium added with interleukin-2 and plasma; and
the immune cell scale amplification culture medium is a serum-free lymphocyte culture medium added with interleukin-2,
the concentration of the sabirine in the immune cell activation culture medium is 0.007-0.013KE/ml,
in the immune cell activation culture medium, the immune cell amplification culture medium and the immune cell scale amplification culture medium, the concentration of the interleukin-2 is 700-1300IU/ml,
in the immune cell activation culture medium, the immune cell amplification culture medium and the immune cell scale amplification culture medium, the serum-free lymphocyte culture medium is: OpTsizerTMCTSTMSerum-free medium or SupercultureTML500 serum-free culture medium for human lymphocyte,
the concentration of the plasma in the immune cell activation medium is 7-13 vol%,
performing the second induced expansion culture when the expression of CD56 by the activated immune cells exceeds 70%;
performing the third scale-up induced expansion culture when the expression of CD56 of the differentiated immune cells exceeds 90%,
the immune cell is a natural killer cell.
2. The method of claim 1, wherein the concentration of sabcomeline in the immune cell activation medium is 0.01 KE/ml.
3. The method of claim 1, wherein the concentration of interleukin-2 in the immune cell activation medium, the immune cell expansion medium, and the immune cell scale-up expansion medium is 1000 IU/ml.
4. The method of claim 1, wherein the concentration of the plasma in the immune cell activation medium is 10% by volume.
5. The method of claim 1, wherein the plasma is autologous plasma.
6. The method of claim 1, wherein the immune cells are passaged every 1 day during the activation of the medium.
7. The method of claim 1, wherein the second induced expansion culture is passaged every 2 days.
8. The method of claim 1, wherein the third induced expansion culture is passaged every 2 days.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611233042.5A CN106754704B (en) | 2016-12-28 | 2016-12-28 | Method for inducing and expanding immune cells in vitro |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611233042.5A CN106754704B (en) | 2016-12-28 | 2016-12-28 | Method for inducing and expanding immune cells in vitro |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106754704A CN106754704A (en) | 2017-05-31 |
| CN106754704B true CN106754704B (en) | 2020-10-16 |
Family
ID=58921395
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611233042.5A Active CN106754704B (en) | 2016-12-28 | 2016-12-28 | Method for inducing and expanding immune cells in vitro |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106754704B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111019892A (en) * | 2019-12-16 | 2020-04-17 | 杭州恩格生物医疗科技有限公司 | Immune cell in-vitro induction amplification method |
| CN111117960A (en) * | 2020-01-09 | 2020-05-08 | 上海奕杉生物科技有限公司 | NK cell in-vitro large-scale induction amplification culture method |
| CN112553157B (en) * | 2020-12-23 | 2023-07-07 | 杭州中赢生物医疗科技有限公司 | Lymphocyte amplification system and method |
| CN114231485A (en) * | 2021-10-29 | 2022-03-25 | 南京国青血液净化科技有限公司 | Immune cell in-vitro induced amplification and preservation method |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101603028A (en) * | 2008-06-10 | 2009-12-16 | 株式会社Nkbio | Medium composition for cultivating self activated lymphocyte |
| CN104593324A (en) * | 2014-11-28 | 2015-05-06 | 广州赛莱拉干细胞科技股份有限公司 | Natural killer cell culture medium and natural killer cell amplification culture method |
| CN105101978A (en) * | 2013-03-27 | 2015-11-25 | 株式会社日本生物治疗研究所 | Method for producing NK cell-enhancing blood product |
| CN105219708A (en) * | 2015-07-21 | 2016-01-06 | 中山大学 | Immunocyte cultivates test kit, immunocyte cultural method and application |
-
2016
- 2016-12-28 CN CN201611233042.5A patent/CN106754704B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101603028A (en) * | 2008-06-10 | 2009-12-16 | 株式会社Nkbio | Medium composition for cultivating self activated lymphocyte |
| CN105101978A (en) * | 2013-03-27 | 2015-11-25 | 株式会社日本生物治疗研究所 | Method for producing NK cell-enhancing blood product |
| CN104593324A (en) * | 2014-11-28 | 2015-05-06 | 广州赛莱拉干细胞科技股份有限公司 | Natural killer cell culture medium and natural killer cell amplification culture method |
| CN105219708A (en) * | 2015-07-21 | 2016-01-06 | 中山大学 | Immunocyte cultivates test kit, immunocyte cultural method and application |
Non-Patent Citations (2)
| Title |
|---|
| OK432-activated natural killer cells enhanced trastuzumab (Herceptin)-mediated antibody-dependent cellular cytotoxicity in patients with advanced cancer;Sudo T et al.;《Anticancer Res.》;20061231;4327-4333 * |
| 两种培养基对细胞因子诱导杀伤细胞体外扩增的影响;张克等;《实用医学杂志》;20111231;第27卷(第22期);4020-4022 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106754704A (en) | 2017-05-31 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108251365B (en) | Immune cell culture medium system | |
| CN106591233B (en) | A kind of external evoked amplification of immunocyte and the method frozen | |
| CN106701681B (en) | A kind of external evoked amplification of immunocyte, the method for freezing and recovering | |
| CN107326008A (en) | A kind of method of high-purity amplifying natural killer cell efficient from peripheral blood | |
| CN111454903B (en) | Immune cell in vitro culture, induction, activation and cryopreservation method and cell bank establishment thereof | |
| CN103756963A (en) | Method used for in vitro proliferation of NK cells | |
| CN108893443A (en) | A kind of Efficient amplification method of cytokine induction umbilical cord blood natural killer | |
| CN106754704B (en) | Method for inducing and expanding immune cells in vitro | |
| CN108588022B (en) | Method for enriching human CD4+ and CD8+ TCM cells through in vitro culture | |
| CN102657853A (en) | Preparation and application of tumor specific killer cells serving as source of initial thymus (T) cells | |
| CN111394309A (en) | Method for in-vitro amplification culture of NK (natural killer) cells | |
| CN108251369B (en) | Immune cell culture medium, culture method and application | |
| CN112608896A (en) | NK cell culture method and application thereof | |
| CN110438077B (en) | Method for simultaneously culturing NK and gamma delta T cells | |
| CN105296421B (en) | The T cell and preparation method of a kind of activation of bispecific antibody and application | |
| CN110747167B (en) | Preparation method and application of hemizygous BAK cell | |
| CN109486758A (en) | A kind of external efficient amplification reagent of peripheral blood NK cell and operating instruction | |
| CN108841790A (en) | A kind of method of the mononuclearcell induction CIK cell in placenta source | |
| CN109957543A (en) | Utilize the method for Cord blood massive amplification Cord Blood Natural Killer Cells: Impact | |
| CN106566807A (en) | Concentration gradient rhIL-2 dependent iNKT cell amplification method and application thereof | |
| CN108192868B (en) | The induced amplification method of immunocyte | |
| CN105535940A (en) | Preparation method of Vgamma9Vdelta2T cell preparation for treating multiple myeloma | |
| CN115896016A (en) | Culture composition and application thereof in culturing immune cells | |
| CN103602634B (en) | The preparation method of DC cell and preparing the application in antitumor cell preparation | |
| CN114480279A (en) | Efficient separation culture technology for human blood immune cells CD4T |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB03 | Change of inventor or designer information | ||
| CB03 | Change of inventor or designer information |
Inventor after: Xi Jiafei Inventor after: Yao Hailei Inventor after: Pei Xuetao Inventor after: Yue Wen Inventor after: Chen Lin Inventor after: Nan Xue Inventor after: Zhang Ya Inventor before: Pei Xuetao Inventor before: Xi Jiafei Inventor before: Yue Wen Inventor before: Chen Lin Inventor before: Yao Hailei Inventor before: Nan Xue Inventor before: Zhang Ya |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20220112 Address after: 100850 No. 27 Taiping Road, Beijing, Haidian District Patentee after: ACADEMY OF MILITARY MEDICAL SCIENCES Patentee after: South China Institute of biomedicine Address before: No.1, helix 4 road, International Biological Island, Guangzhou, Guangdong 510200 Patentee before: SOUTH CHINA INSTITUTE OF BIOMEDICINE |